Methods for treating b-cell lymphoma by administering an anti-cd20 antibody

ABSTRACT

The present invention provides methods for treating a B-cell lymphoma in a human subject. The methods of the invention comprise administering to a subject in need thereof an antibody or antigen-binding fragment thereof that specifically binds human CD20. In certain embodiments, the methods of the invention are useful for treating non-Hodgkin&#39;s B-cell lymphoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/965,956, filed on Dec. 13, 2010, which is a divisional of U.S.application Ser. No. 12/183,274, filed on Jul. 31, 2008, now U.S. Pat.No. 7,879,984, which claims the benefit under 35 USC §119(e) of U.S.Provisional Appl. Nos. 60/962,811 filed on Jul. 31, 2007, and 61/067,994filed Mar. 3, 2008, which applications are herein specificallyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention related to human antibodies and antibody fragmentsspecific for human CD20, pharmaceutical compositions, and therapeuticmethods thereof.

STATEMENT OF RELATED ART

CD20 (also known as human B-lymphocyte-restricted differentiationantigen or Bp35; B-lymphocyte surface antigen B1, Leu-16, BMS, and LF5)is a hydrophobic transmembrane protein with a molecular weight of ˜35 kDexpressed on pre-B and mature B lymphocytes (Valentine et al. (1989) JBiol Chem 264:11282; Einfield et al. (1988) EMBO J 7:711-717). The aminoacid sequence of human CD20 is shown in SEQ ID NO:1 (GenBank AccessionNo. NP_(—)690605). Anti-CD20 antibodies are described in, for example,U.S. Pat. No. 5,736,137, WO 2004/056312, and US 2004/0167319, whichpublications are herein specifically incorporated by reference in theirentirety.

Methods for producing antibodies useful as human therapeutics includegeneration of chimeric antibodies and humanized antibodies (see, forexample, U.S. Pat. No. 6,949,245). See also, for example, WO 94/02602and U.S. Pat. No. 6,596,541 (both of which publications are hereinspecifically incorporated by reference) describing methods of generatinggenetically modified mice capable of producing antibodies useful formaking human therapeutics.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides human antibodies, preferablyrecombinant human antibodies that specifically bind human CD20. Theseantibodies are characterized by specifically binding to human CD20 andby mediating the killing of B-cell lymphoma cells expressing CD20. Theantibodies can be full-length (for example, an IgG1 or IgG4 antibody) ormay comprise only an antigen-binding portion (for example, an Fab,F(ab′)₂ or scFv fragment), and may be modified to effect functionality,e.g., to eliminate or enhance residual effector functions (Reddy et al.(2000) J. Immunol. 164:1925-1933).

The antibody or an antigen-binding fragment thereof specifically bindshuman CD20 and is capable of inducing complement dependent cytotoxicity(CDC) of cells expressing CD20 in the presence of complement, whereinthe antibody at a concentration of about 10 nM or less induces 50% lysisof Daudi and RL cells in the presence of 5% normal human serum withcomplement. In preferred embodiments, the antibody concentration whichinduces 50% lysis is about 5 nM or less; about 2 nM or less; or about 1nM or less. In one embodiment, the antibody or fragment thereofexhibiting an EC₅₀ 0.2 nM or less as measured in Daudi cells, or an EC₅₀of 0.4 nM or less as measured by RL cells. In various embodiments, theantibody or antibody fragment is capable of increasing symptom freesurvival time between about 2-fold to about 9-fold or more, relative tocontrol-treated animals in a mouse model of human lymphoma.

The antibody or fragment thereof specifically binds human CD20 and iscapable of inducing antibody-dependent cellular cytotoxicity (ADCC) ofcells expressing CD20 in the presence of peripheral blood mononuclearcells (PBMC), wherein the antibody exhibits an EC₅₀ of about 1 nM orless, as measured in Daudi cells. In preferred embodiments, the antibodyexhibits an EC₅₀ of about 50 pM or less; about 20 pM or less; about 10pM or less. In a preferred embodiment, antibodies exhibits enhanced ADCCactivity may comprise reduced levels of fucosylation, for example, about5% fucose.

In one embodiment, the antibody or antigen-binding portion of theantibody of the invention comprises a heavy chain variable region (HCVR)sequence selected from the group consisting of SEQ ID NO:3, 19, 23, 27,43, 47, 51, 67, 71, 75, 91, 95, 99, 115, 119, 123, 139, 143, 147, 163,167, 171, 187, 191, 195, 211, 215, 219, 235, 239, 243, 259, 263, 267,283, 287, 291, 307, 311, 315, 331, 335, 339, 355, 359, 363, 379, 383,387, 395, and 403, or a substantially similar sequence thereof. In apreferred embodiment, the antibody or fragment comprises a HCVR sequenceselected from the group consisting of SEQ ID NO:339, 195 and 243.

In a more specific embodiment, the antibody or antigen-binding fragmentthereof further comprises a light chain variable region (LCVR) sequenceselected from the group consisting of SEQ ID NO:11, 21, 25, 35, 45, 49,59, 69, 73, 83, 93, 97, 107, 117, 121, 131, 141, 145, 155, 165, 169,179, 189, 193, 203, 213, 217, 227, 237, 241, 251, 261, 265, 275, 285,289, 299, 309, 313, 323, 333, 337, 347, 357, 361, 371, 381 and 385, or asubstantially similar sequence thereof. In a preferred embodiment, theantibody or fragment comprises a LCVR selected from the group consistingof SEQ ID NO:347, 203 and 251.

In specific embodiments, the antibody or fragment thereof comprisesHCVR/LCVR sequence pairs selected from the group consisting of SEQ IDNO:3/11, 19/21, 23/25, 27/35, 43/45, 47/49, 51/59, 67/69, 71/73, 75/83,91/93, 95/97, 99/107, 115/117, 119/121, 123/131, 139/141, 143/145,147/155, 163/165, 167/169, 171/179, 187/189, 191/193, 195/203, 211/213,215/217, 219/227, 235/237, 239/241, 243/251, 259/261, 263/265, 267/275,283/285, 287/289, 291/299, 307/309, 311/313, 315/323, 331/333, 335/337,339/347, 355/357, 359/361, 363/371, 379/381 and 383/385. In a preferredembodiment, the antibody or fragment thereof comprises HCVR/LCVRsequence pair selected from the group consisting of SEQ ID NO:339/347,195/203 and 243/251.

In a second aspect, the invention provides isolated nucleic acidmolecules that encode an antibody or fragment thereof. In specificembodiments, the nucleic acid molecule encodes an HCVR wherein thenucleotide sequence is selected from the group consisting of SEQ IDNO:2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122,138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238,242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354,358, 362, 378, 382, 386, 394 and 402, or a substantially identicalsequence thereof. In a related aspect, the invention provides anisolated nucleic acid molecule encoding an LCVR, wherein the nucleotidesequence is selected from the group consisting of SEQ ID NO: 10, 20, 24,34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154,164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, 264,274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380 and384, or a substantially identical sequence thereof. In a preferredembodiment, the antibody or antibody fragment comprise an HCVR encodedby a nucleic acid molecule selected from the group consisting of SEQ IDNO:338, 194 and 242, and a LCVR encoded by a nucleic acid moleculeselected from the group consisting of SEQ ID NO:346, 202 and 250,respectively.

In a third aspect, the invention features an antibody or antigen-bindingfragment thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 9,33, 57, 81, 104, 129, 153, 177, 201, 225, 249, 273, 297, 321, 345, 369,393, 401 and 409; and a light chain CDR3 (LCDR3) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:17, 41,65, 89, 113, 137, 161, 185, 209, 233, 257, 281, 305, 329, 353 and 377.In a preferred embodiment, the antibody or fragment thereof comprises aHCDR3 and LCDR3 sequence selected from the HCDR3/LCDR3 sequence pairsSEQ ID NO: 345/353, 201/209 and 249/257.

In a more specific embodiment, the antibody or fragment thereof furthercomprises a heavy chain CDR1 (HCDR1) domain sequence selected from thegroup consisting of SEQ ID NO:5, 29, 53, 77, 101, 125, 149, 173, 197,221, 245, 269, 293, 317, 341, 365, 389, 397 and 405; a heavy chain CDR2(HCDR2) domain sequence selected from the group consisting of SEQ IDNO:7, 31, 55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343,367, 391, 399 and 407; a light chain CDR1 (LCDR1) domain sequenceselected from the group consisting of SEQ ID NO:13, 37, 61, 85, 109,133, 157, 181, 205, 229, 253, 277, 301, 325, 349 and 373; and a lightchain CDR2 (LCDR2) domain sequence selected from the group consisting ofSEQ ID NO:15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303,327, 351 and 375. In a preferred embodiment, the antibody or fragmentthereof comprises heavy and light chain CDRs sequences selected from thegroup consisting of SEQ ID NO:341, 343, 345, 349, 351 and 353; 197, 199,201, 205, 207 and 209; and 245, 247, 249, 253, 255 and 257,respectively.

In a fourth aspect, the invention features isolated nucleic acidmolecules encoding an antibody or antigen-binding fragments of theinvention, wherein the nucleic acid molecules encoding a HCDR3 domainand a LCDR3 domain are selected from the group consisting of SEQ ID NO:9and 16; 33 and 41; 57 and 65; 81 and 89; 104 and 113; 129 and 137; 153and 161; 177 and 185; 201 and 209; 225 and 233; 249 and 257; 273 and281; 297 and 305; 321 and 329; 345 and 353; and 369 and 377,respectively.

In a fifth aspect, the invention features an antibody or antigen-bindingfragment, comprising a HCDR3 domain and a LCDR3 domain, wherein theHCDR3 domain comprises an amino acid sequence of the formulaX¹-X²-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶-X¹⁷-X¹⁸-X¹⁹ (SEQ IDNO:412) wherein X¹=A, V or T; X²=K; X³=D; X⁴=P, F or G; X⁵=S or H; X⁶=Y;X²=G; X⁸=S or H; X⁹=G or F; X¹⁰=S or Y; X¹¹=Y, N or S; X¹²=Y, G or H;X¹³=G, L or S; X¹⁴=Y, M or D; X¹⁵=Y, D or V; X¹⁶=G, V or absent; X¹⁷=Mor absent; X¹⁸=D or absent; X¹⁹=V or absent; and the LCDR3 domaincomprises an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:415), wherein X¹=Q; X²=Q; X³=R orS; X⁴=N, Y or F; X⁵=N, D, or Y; X⁶=W; X²=P; X⁸=L; X⁹=T.

In a more specific embodiment, the antibody or antigen-binding fragmentfurther comprises heavy and light chain CDR1 and CDR2 domains, whereinthe HCDR1 domain comprises an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:410) wherein X¹=G; X²=F or I; X³=T;X⁴=F; X⁵=H, R or Y; X⁶=D; X⁷=Y; X⁸=T or A; the HCDR2 domain comprises anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ IDNO:411) wherein X¹=I; X²=S; X³=W; X⁴=N; X⁵=S; X⁶=G or D; X⁷=S, Y or T;X⁸=I or L; the LCDR1 domain comprises an amino acid sequence of theformula X¹-X²-X³-X⁴-X⁵-X⁶ (SEQ ID NO:413) wherein X¹=Q; X²=S; X³=V or I;X⁴=S; X⁵=S or R; X⁶=Y or N; and the LCDR2 domain comprises an amino acidsequence of the formula X¹-X²-X³ (SEQ ID NO:414) wherein X¹=E, G or V;X²=A; X³=S.

In a sixth aspect, the invention provides recombinant expression vectorscarrying the nucleic acid molecules of the invention, and host cellsinto which such vectors have been introduced, as well as methods ofmaking the antibodies or fragments thereof of the invention obtained byculturing the host cells of the invention. The host cell may be aprokaryotic or eukaryotic cell, preferably the host cell is an E. colicell or a mammalian cell, such as a CHO cell. In a preferred embodiment,an antibody may be produced with varying amounts of fucosylation. Forexample, a CHO cell line may be selected to produce an antibody orantibody fragment with a range of fucosylation from a minimum of about5% to a maximum of about 95%.

In a seventh aspect, the invention features a pharmaceutical compositioncomprising a anti- human CD20 antibody or fragment thereof and apharmaceutically acceptable carrier.

In an eighth aspect, the invention features a fully human antibody orantibody fragment capable of binding to human CD20, with a EC₅₀ of lessthan about 10 nM, as measured by cell binding experiments (describedbelow). In a preferred embodiment, the antibody of the inventionexhibits an EC₅₀ of about 10⁻⁹ to about 10⁻¹² M or higher, for example,at least 10⁻⁹ M, at least 10⁻⁹ M, at least 10¹⁰ M, at least 10⁻¹¹ M, orat least 10⁻¹² M, when measured by binding to antigen presented on cellsurface.

The invention encompasses anti-CD20 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof a galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In a ninth aspect, the invention features methods for inhibiting CD20activity using an antibody, or fragment thereof. In one embodiment, themethod comprises administering a therapeutically effective amount of ananti-CD20 antibody or antibody fragment to a human subject sufferingfrom, for example, non-Hodgkin's lymphoma, rheumatoid arthritis,systemic lupus erythematosus, Crohn's disease, chronic lymphocyticleukemia, and inflammatory diseases.

Other objects and advantages will become apparent from a review of theensuing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Symptom-free survival curve. Results are shown for human Fccontrol, control antibodies I and II, and antibodies: 8G6-5, 9D4-7,10F2-13, and 7E1-13.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “CD20” includes variants and isoforms of human CD20, which arenaturally expressed by cells. Binding of an antibody of the invention tothe CD20 antigen mediates the killing of cells expressing CD20 (forexample, a tumor cell). The killing of cells expressing CD20 may occurin a number of ways, including complement dependent cytotoxicity (CDC)of cells expressing CD20, apoptosis of cells expressing CD20, effectorcell phagocytosis of cells expressing CD20, or effector cell antibodydependent cellular cytotoxicity (ADCC) of cells expressing CD20.

The term “antibody,” as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain comprises a heavy chain variable region (abbreviatedherein as HCVR or VH) and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion comprises of one domain (CL1). The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementary determining regions (CDR), interspersed with regions thatare more conserved, termed framework regions (FR). Each VH and VL iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion” or “antibody fragment”), as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., hCD20). It has been shown that fragments of afull-length antibody can perform the antigen-binding function of anantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) an Fab fragment, amonovalent fragment consisting of the VL, VH, CL1 and CH1 domains; (ii)an F(ab′)₂ fragment, a bivalent fragment comprising two F(ab)′ fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH1 domains; (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (v) a dAb fragment(Ward et al. (1989) Nature 241:544-546), which consists of a VH domain;and (vi) an isolated complementary determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singlecontiguous chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies, are alsoencompassed (see e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA90:6444-6448).

A “CDR” or complementary determining region is a region ofhypervariability interspersed within regions that are more conserved,termed “framework regions” (FR). In various embodiments of theanti-hCD20 antibody or fragment of the invention, the FRs may beidentical to the human germline sequences, or may be naturally orartificially modified.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB).

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Epitopes may be either conformational or linear. Aconformational epitope is produced by spatially juxtaposed amino acidsfrom different segments of one (or more) linear polypeptide chain(s). Alinear epitope is an epitope produced by adjacent amino acid residues ina polypeptide chain. In certain circumstance, an epitope may includeother moieties, such as saccharides, phosphoryl groups, or sufonylgroups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor GAP, as discussed below.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions, whichare not identical, differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent or degree of similarity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, herein incorporated by reference. Examples of groups of aminoacids that have side chains with similar chemical properties include 1)aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443 45, herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 andAltschul et al. (1997) Nucleic Acids Res. 25:3389 402, each of which isherein incorporated by reference.

The term “effective amount” is a concentration or amount of an antibodyor antigen-binding fragment of an antibody which results in achieving aparticular stated purpose. An “effective amount” of an anti-CD20antibody or antigen-binding fragment of an antibody thereof may bedetermined empirically. Furthermore, a “therapeutically effectiveamount” is a concentration or amount of an anti-CD20 antibody orantigen-binding fragment thereof which is effective for achieving astated therapeutic effect This amount may also be determinedempirically.

Preparation of Human Antibodies

Methods for generating human antibodies include, for example,VelocImmune™ (Regeneron Pharmaceuticals), XenoMouse™ technology (Greenet al. (1994) Nature Genetics 7:13-21; Abgenix), the “minilocus”approach, and phage display (and see, for example, U.S. Pat. No.5,545,807, U.S. Pat. No. 6,787,637). VelocImmune™ technology (U.S. Pat.No. 6, 596,541) encompasses a method of generating a high specificityfully human antibody to a select antigen. This technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody. In specific embodiment, the cell is a CHO cell.

Antibodies may be therapeutically useful in blocking a ligand-receptorinteraction or inhibiting receptor component interaction, rather than bykilling cells through fixation of complement (CDC) and participationantibody-dependent cell-mediated cytotoxicity (ADCC). The constantregion of an antibody is important in the ability of an antibody to fixcomplement and mediate cell-dependent cytotoxicity. Thus, the isotype ofan antibody may be selected on the basis of whether it is desirable forthe antibody to mediate cytotoxicity.

Human immunoglobulins can exist in two forms that are associated withhinge heterogeneity. In one form, an immunoglobulin molecule comprises astable four-chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via interchain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification. Thefrequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30: 105) to levels typically observed usinga human IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, CH2 or CH3 region, which may bedesirable, for example, in production, to improve the yield of thedesired antibody, form.

Antibodies of the invention are preferably prepared with the use ofVelocImmune™ technology. A transgenic mouse in which the endogenousimmunoglobulin heavy and light chain variable regions are replaced withthe corresponding human variable regions is challenged with the antigenof interest, and lymphatic cells (such as B-cells) are recovered fromthe mice that express antibodies. The lymphatic cells may be fused witha myeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) or EC₅₀ of from about 10⁻⁸through about 10⁻¹² M or higher, for example, at least 10⁻⁸ M, at least10⁻⁹ M, at least 10⁻¹⁰ M, at least 10⁻¹¹ M, or at least 10⁻¹² M, whenmeasured by binding to antigen presented on cell surface.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As described below, theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatethe fully human antibody of the invention, for example wild-type ormodified IgG1 or IgG4 (for example, SEQ ID NO:416, 417, 418). While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

Epitope Mapping and Related Technologies

To screen for antibodies which bind to a particular epitope, a routinecross-blocking assay such as that described in “Antibodies: A LaboratoryManual” 1988 Cold Spring Harbor Laboratory, Harlow and Lane, eds.(herein specifically incorporated by reference in its entirety) can beperformed. Other methods include alanine scanning mutants, peptide blots(Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavageanalysis as described in the examples below. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer (2000) Protein Science: 9: 487-496).

To ascertain the binding characteristics of the antibodies, mutant CD20proteins consisting of selected amino acid substitutions wereconstructed. The mutant CD20 proteins contained substitutions of certainamino acids occurring in the human protein with corresponding aminoacids occurring in the mouse protein. This approach helped ensure thatthe mutant CD20 proteins maintained their tertiary structure and,presumably, any conformational epitopes. Binding of the test antibodiesto these mutant CD20 proteins was compared with binding of control(known) CD20 antibodies, as measured by FACS. None of the inventiveantibodies displayed a binding profile that was identical (with respectto each and every mutant) to either of the control antibodies.

Immunoconjugates

The invention encompasses a human anti-CD20 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example, WO05/103081, herein specifically incorporated by reference in itsentirety).

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-CD20 antibodies canbe linked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment, to produce abispecific or a multispecific antibody with a second bindingspecificity. A multispecific antibody of the invention may specificallybind both a CD20 expressing cell and a human effector cell expressing apolypeptide, such as a human Fc receptor, and/or components of the Tcell receptor complex. In one embodiment, the multispecific antibody ofthe invention comprises a CD20-binding portion and a cytokine.

Therapeutic Uses

The human antibodies, antigen-binding fragments of antibodies,immunoconjugates, and bispecific molecules of the invention are usefulin therapeutic methods for treating human diseases which are inhibitedor ameliorated by inhibiting growth of cells expressing CD20 and/orkilling cells expressing CD20. The mechanism of action by which thetherapeutic methods of the invention are achieved include killing of thecell expressing CD20 in the presence of effector cells, for example, byCDC, apoptosis, ADCC, phagocytosis, or by a combination of two or moreof these mechanisms. The mechanism for achieving the therapeutic effectof the molecules of the invention may result in direct killing orinhibition of cells expressing CD20, or indirectly, through inhibitingcells which do not express CD20 for, for example, express a structurallyrelated cell-surface antigen (i.e., without cross-reactivity to relatedbut functionally distinct cell surface antigens). Cells expressing CD20which can be inhibited or killed using the human antibodies of theinvention include, for example, tumorigenic B cells.

Examples of diseases and conditions that can be treated or amelioratedwith the anti-CD20 antibodies and fragments thereof include, but are notlimited to, tumorigenic diseases, such as B cell lymphoma (NHL,precursor B cell lymphoblastic leukemia/lymphoma, mature B cellneoplasms, B cell chronic lymphocytic leukemia/small lymphocyticlymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma, follicular lymphoma, cutaneous follicle centerlymphoma, marginal zone B cell lymphoma, hairy cell leukemia, diffuselarge B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cellmyeloma, post-transplant lymphoproliferative disorder, Waldenstrom'smacroglobulinemia, and anaplastic large-cell lymphoma); immune diseases,such as autoimmune diseases (psoriasis, psoriatic arthritis, dermatitis,systemic scleroderma and sclerosis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, respiratory distress syndrome, meningitis,encephalitis, uveitis, glomerulonephritis, eczema, asthma,atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis,Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes,Reiter's disease, Behcet's disease, immune complex nephritis, IgAnephropathy, IgM polyneuropathies); immune-mediated thrombocytopenias,acute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupusnephritis, systemic lupus erythematosus, rheumatoid arthritis, atopicdermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis,Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acuteinfectious mononucleosis, HIV, and herpes virus associated diseases;severe acute respiratory distress syndrome and choreoretinitis; diseasesand disorders caused by infection of B-cells with virus, such asEpstein-Barr virus.

In a specific embodiment, the subject being administered the antibody isadditionally treated with a chemotherapeutic agent, radiation, or anagent that modulates (enhances or inhibits) the expression or activityof an Fc receptor, such as a cytokine. Typical, cytokines foradministration during treatment include granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), interferon-gamma. (IFN-γ), and tumor necrosis factor (TNF).Typical therapeutic agents include, among others, anti-neoplastic agentssuch as doxorubicin, cisplatin, bleomycin, carmustine, chlorambucil, andcyclophosphamide.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the humananti-CD20 antibodies or antigen-binding fragments thereof of the presentinvention. The therapeutic compositions in accordance with the inventionwill be administered with suitable carriers, excipients, and otheragents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. In general, carriers,excipients, or other agents can include, for example, oils (e.g.,canola, cottonseed, peanut, safflower, sesame, soybean), fatty acids andsalts and esters thereof (e.g., oleic acid, stearic acid, palmiticacid), alcohols (e.g., ethanol, benzyl alcohol), polyalcohols (e.g.,glycerol, propylene glycols and polyethylene glycols, e.g., PEG 3350),polysorbates (e.g., polysorbate 20, polysorbate 80), gelatin, albumin(e.g., human serum albumin), salts (e.g., sodium chloride), succinicacid and salts thereof (e.g., sodium succinate), amino acids and saltsthereof (e.g., alanine, histidine, glycine, arginine, lysine), aceticacid or a salt or ester thereof (e.g., sodium acetate, ammoniumacetate), citric acid and salts thereof (e.g., sodium citrate), benzoicacid and salts thereof, phosphoric acid and salts thereof (e.g.,monobasic sodium phosphate, dibasic sodium phosphate), lactic acid andsalts thereof, polylactic acid, glutamic acid and salts thereof (e.g.,sodium glutamate), calcium and salts thereof (e.g., calcium chloride,calcium acetate), phenol, sugars (e.g., glucose, sucrose, lactose,maltose, trehalose), erythritol, arabitol, isomalt, lactitol, maltitol,mannitol, sorbitol, xylitol, nonionic surfactants (e.g., TWEEN® 20,TWEEN® 80), ionic surfactants (e.g., sodium dodecyl sulfate),chlorobutanol, DMSO, sodium hydroxide, glycerin, m-cresol, imidazole,protamine, zinc and salts thereof (e.g, zinc sulfate), thimerosal,methylparaben, propylparaben, carboxymethylcellulose, chlorobutanol, andheparin. Other non-therapeutic agents are described in U.S. Pat. No.7,001,892, incorporated herein by reference, in particular in Table A. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences (Mack Publishing Company, Easton, Pa). These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. Any of the foregoing mixtures may be appropriate intreatments and therapies in accordance with the present invention,provided that the active ingredient in the formulation is notinactivated by the formulation and the formulation is physiologicallycompatible and tolerable with the route of administration. See alsoPowell et al. PDA (1998) J Pharm Sci Technol. 52:238-311 and thecitations therein for additional information related to excipients andcarriers well known to pharmaceutical chemists.

The dose of the therapeutic compositions may vary depending upon the ageand the size of a subject to be administered, target disease,conditions, route of administration, and the like. When the antibody ofthe present invention is used for treating various conditions anddiseases associated with CD20 activity, including non-Hodgkin'slymphoma, rheumatoid arthritis, systemic lupus erythematosus, Crohn'sdisease, chronic lymphocytic leukemia, inflammatory diseases, and thelike, in an adult patient, it is advantageous to intravenouslyadminister the antibody of the present invention normally at a singledose of about 0.01 to about 20 mg/kg body weight, preferably about 0.1to about 10 mg/kg body weight, and more preferably about 0.1 to about 5mg/kg body weight. Depending on the severity of the condition ordisease, the frequency and the duration of the treatment can beadjusted. In other parenteral administration and oral administration,the antibody can be administered in a dose corresponding to the dosegiven above.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration may preferably be systemic orlocal.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533). Incertain situations, the pharmaceutical composition can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). Inanother embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984).In yet another embodiment, a controlled release system can be placed inproximity of the composition's target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138, 1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the aforesaidantibody is contained in about 5 to 100 mg and in about 10 to 250 mg forthe other dosage forms.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human CD20

Immunization of rodents can be done by any method known in the art (see,for example, Harlow & Lane, eds. (1988) Antibodies: A Laboratory Manual,Cold Spring Harbor Press, New York; Malik and Lillehoj, Antibodytechniques: Academic Press, 1994, San Diego). In one embodiment, cellsexpressing CD20 are administered directly to mice which have DNA lociencoding both human Ig heavy chain variable regions and Kappa lightchain variable regions (VelocImmune™, Regeneron Pharmaceuticals, Inc.;U.S. Pat. No. 6,596,541), with an adjuvant to stimulate the immuneresponse. Such an adjuvant can include complete and incomplete Freund'sadjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides)(see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, Totawa, N.J.). Toachieve high expression levels of human CD20 on a cell surface, themurine cell lines, MG87 and/or NS/0 cells, are transfected with aplasmid encoding for human CD20, and cells expressing high levels ofCD20 are enriched using FACS technology. In one embodiment, CD20 isadministered indirectly as a DNA plasmid that contains a CD20 gene, andCD20 is expressed using the host's protein expression system to produceantigen protein in vivo. In both approaches, to attain optimal antibodyimmune response, mice are given booster injections every 3-4 weeks. Theimmune response is monitored by a cell-based immunoassay as describedbelow in which serum samples in 1- to 3-fold serial dilutions areimmunoassayed. Serum titer is defined as the dilution of serum samplethat yielded an assay signal two-fold over background. When animalsreach their maximum immune response, antibody-expressing B cells areharvested and fused with mouse myeloma cells to form hybridomas.

Example 2 Screening for Antigen Specific Hybridoma

In primary screening, NS/0 cells (ATCC) were transfected with the humanCD20 gene and high-expressing cells (NS/0-hCD20 cells) were pooled andmaintained in culture for use in screening hybridoma-conditioned media,generally about 11 to 14 days following fusion. NS/0-hCD20 cells in RPMI1640 with 10% fetal calf serum were plated at a density of 50,000 cellsper well in 96-well poly-D-lysine plates. Hybridoma-conditioned mediumwas diluted 5-fold and allowed to bind to the cells for 30 minutes. Thecells were then fixed onto the plates with the addition of an equalvolume of 8% formaldehyde for 20 min, followed by four successive PBSTwashes. The plates were incubated with 5% BSA for 2 hrs at roomtemperature (RT). After washing, plate-bound antibodies were incubatedwith HRP-conjugated goat anti-mouse IgG Fcy-specific polyclonalantibodies for 30 min, and the plates developed using 3.3′,5.5′-tetramethyl-benzidine (TMB) substrate (BD Pharmigen) following thefinal washes. The HRP reaction was stopped with an equal volume of 1 Mphosphoric acid. Antibody binding signals were measured by opticaldensity at 450 nm. NS/0 parental cells, which have no detectable CD20expression, were used as a background control to exclude hybridomasupernatants with non-specific cell surface binding. Wells positive forboth NS/0 parental cells and CD20-expressing cells were excluded.

Example 3 Sequencing of Human Antibodies Against CD20

Prior to sequencing, antigen-specific hybridoma cells were single-cellsub-cloned using a MOFLO™ flow cytometer. Sequencing of the variablelight and heavy chain regions was performed by standard methods (see forexample, US 2004/0167319A1, herein specifically incorporated byreference in its entirety). Total RNA was prepared from each hybridomacell line with an RNEASY™ kit (Qiagen). cDNA was prepared using theSMART RACE™ cDNA Amplification kit (Clonetech). DNA sequences of HCVRsand LCVR were sequenced and the predicted amino acid sequences for HCVRsand LCVRs provided for selected antibodies (HCVR/LCVR SEQ ID NO):3B9-10N (3/11); 3B9-10GSP (19/21); 3B9-10FGL (23/25); 9C11-14N (27/35);9C11-14GSP (43/45); 9C11-14FGL (47/49); 2B7-7N (51/59); 2B7-7GSP(67/69); 2B7-7FGL (71/73); 2C11-4N (75/83); 2C11-4GSP (91/93); 2C11-4FGL(95/97); 3H7-6N (99/107); 3H7-6GSP (115/117); 3H7-6FGL (119/121);5H2-17N (123/131); 5H2-17GSP (139/141); 5H2-17FGL (143/145); 6B9-4N(147/155); 6B9-4GSP (163/165); 6B9-4FGL (167/169); 6F6-1N (171/179);6F6-1GSP (187/189); 6F6-1FGL (191/193); 8G6-5N (”8G6-5″) (195/203);8G6-5GSP (211/213); 8G6-5FGL (215/217); 9C3-8N (219/227); 9C3-8GSP(235/237); 9C3-8FGL (239/241); 9D4-7N (“9D4-7”) (243/251); 9D4-7GSP(259/261); 9D4-7FGL (263/265); 9E4-20N (267/275); 9E4-20GSP (283/285);9E4-20FGL (287/289); 9H4-12N (291/299); 9H4-12GSP (307/309); 9H4-12FGL(311/313); 10E3-17N (315/323); 10E3-17GSP (331/333); 10E3-17FGL(335/337); 10F2-13N (“10F2-13”) (339/347); 10F2-13GSP (355/357);10F2-13FGL (359/361); 7E1-13N (363/371); 7E1-13GSP (379/381); 7E1-13FGL(383/385).

Example 4 Antigen Binding Specificity of the Anti-CD20 Antibodies

After chimeric antibodies had been converted to fully human IgGs,specific antigen binding properties were determined with an ELISAprotocol similar to the protocol described above, except that anHRP-conjugated goat anti-hIgG Fcγ-specific polyclonal was used as thedetection antibody and a Daudi cell line (which expresses endogenousCD20) was used as an antigen source. All of the tested antibodies boundspecifically to Daudi cells with EC₅₀ values ranging from about 0.4 nMto about 20 nM.

Antigen binding specificity of the fully human anti-CD20 antibodies wasverified using flow cytometry as described below, with humanCD20-transfected MG87 cells. Briefly, parental MG87 and humanCD20-transfected MG87 cells were incubated for 30 min at 4° C. with eachof the 15 human antibodies and the two control antibodies, followed byincubation with PE-conjugated anti-human IgG antibody. Binding wasassessed by flow cytometry. Fluorescence intensities were compared withbinding to the parental cell line and control isotype-matched sample.Results are summarized on Table 1. All antibodies bound to humanCD20-transfected MG87 cells, whereas no binding was observed to parentalMG87 cells, indicating that the antibodies are CD20-specific. Control I:chimeric (murine/human) anti-CD20 mAb, rituximab, (RITUXAN®, IDECPharmaceuticals Corp.); control II: human anti-CD20 mAb, 2F2, describedin WO 2005/103081).

TABLE 1 Total Mean Fluorescent Intensity Antibody Untransfected HumanCD20-Transfected Unstained 5.78 5.86 Control I 6.15 2955.71 Control II6.08 3315.94 7E1-13 6.11 3076.88 2B7-7 6.08 3483.32 10F2-13 6.13 3396.699H4-12 6.06 2043.95 10E3-17 6.03 3071.01 9D4-7 6.66 3156.91 9C3-8 6.032913.87 3B9-10 6.07 2986.32 9E4-20 6.03 2908.67 3H7-6 6.1 3302.01 6B9-46.09 2933.36 6F6-1 6.05 3385.59 8G6-5 6.04 3407.87 2C11-4 6.02 2009.869C11-14 6.05 2751.56

Example 5 Human Anti-CD20 Antibody Binding to Mutant Human CD20

Mutant human CD20s were generated by substituting human CD20 amino acidsequences with corresponding mouse amino acids using a StrategeneMutagenesis kit (Table 2). A plasmid vector comprising a mutant humanCD20, a CMV promoter, and a hygromycin resistant gene-IRES-GFP markerwas then transfected into MG87 cells. For each mutant human CD20, a poolof hygromycin-resistant cells that displayed high GFP expression werecollected and a stable line was created for antibody binding assay.

TABLE 2 Mutant Mutation(s) #1 Y77F #2 N163D #3 A170S P172S #4 N166D #5P172S #6 Y77F N166D #7 Y77F N163D #8 Y77F N163D N166D #9 N163D N166D #10A157V

Briefly, approximately 1×10⁶ cells from each stably transfected cellline expressing a mutant human CD20 were collected and incubated witheach anti-human antibody, at 10 μg/ml, on ice for 1 hr, followed byincubation with APC-conjugated goat anti-human IgG (Jackson Immunolabs),at 10 μg/ml, on ice for 45 min. For each antibody, binding to eachmutant human CD20 was assessed by flow cytometry. Mean fluorescenceintensity levels were assessed while gating on a small (approximately20%) population of cells that displayed a median level of GFP expressionto minimize effects due to variable mutant CD20 expression levels withineach cell line. For each mutant CD20, the antibody that displayed thehighest mean fluorescence intensity was designated as 100% binding.Table 3 shows the percent binding of each anti-CD20 antibody to eachmutant human CD20.

TABLE 3 Percent of Binding to Mutant Human CD20 (%) Antibody #1 #2 #3 #4#5 #6 #7 #8 #9 #10 Control II 60 30 70 51 84 1 1 0 4 79 3B9-10 56 6 6740 72 1 1 0 4 79 9C11-14 89 83 0 87 29 87 96 100 100 83 7E1-13 94 49 9584 85 1 1 0 4 92 6F6-1 100 74 100 85 100 16 2 0 4 98 8G6-5 86 45 65 7076 5 1 0 4 96 10F2-13 79 54 55 60 69 1 2 0 4 87 2B7-7 100 40 87 100 8812 2 0 4 83 10E3-17 61 1 6 1 49 1 1 0 4 87 2C11-4 35 0 11 31 30 1 1 0 368 9D4-7 67 2 70 5 66 0 2 0 4 75 6B9-4 74 3 64 3 67 1 2 0 3 75 3H7-6 281 43 4 45 0 1 0 3 80 9C3-8 36 0 84 22 76 0 1 0 3 77 9H4-12 19 0 0 0 2 12 0 4 52 9E4-20 14 0 57 8 57 1 1 0 3 76 Control I 96 100 0 96 2 100 10093 97 100

Example 6 Potency in Complement Dependent Cytotoxicity (CDC)

The anti-human CD20 human antibodies were tested for their ability topromote complement dependent cytotoxicity (CDC) using the human lymphomacell lines Daudi and RL as target cell lines. The antibodies wereserially diluted (final concentration range of 50 nM to 0.85 pM plusbuffer control) into media and added to target cells seeded in a 96 wellplate format. Human serum with complement components (Quidel) was addedto each well to give a final serum concentration of 5%. The cells wereincubated at 37° C. for 2 hrs with the test antibodies and human serumwith complement components and then assayed for cell survival asdetected by ALAMARBLUE™. Fluorescence was measured using an excitationwavelength of 560 nm and an emission wavelength of 590 nm (Table 4).

TABLE 4 Antibody Daudi EC₅₀ (nM) n RL EC₅₀ (nM) N 10F2-13 0.17 ± 0.08 30.36 ± 0.10 4 8G6-5 0.21 ± 0.08 3 1.06 ± 0.43 4 9D4-7 0.22 ± 0.21 4 0.83± 0.60 5 2B7-7 0.24 ± 0.09 4 1.03 ± 0.40 5 Control II 0.28 ± 0.11 5 0.77± 0.41 6 6B9-4 0.34 ± 0.25 3 0.97 ± 0.32 4 3H7-6 0.44 ± 0.27 2 3.66 ±3.85 2 6F6-1 0.56 ± 0.35 3 1.20 ± 0.43 4 10E3-17 0.59 ± 0.24 2 7.80 ±8.64 3 Control I 0.84 ± 0.60 6 >50 4 9E4-20 1.53 ± 0.87 3 1.70 ± 1.80 47E1-13 1.59 ± 0.71 3 5.81 ± 3.77 4 3B9-10 1.86 ± 0.96 3 8.84 ± 6.94 49C3-8 2.22 ± 1.62 2 11.13 ± 9.29  2 9C11-14 7.14 ± 6.63 3 12.01 ± 6.61 4 9H4-12 51.10 ± 38.4  2 29.60 ± 23.76 2 2C11-4 >50 2 5.19 ± 3.10 3

Example 7 Functional Off-Rate of Human Anti-CD20 Antibodies

The off-rates of the anti-CD20 mAbs were analyzed in a CDC assay. Theexperiments were performed in 3 separate sets. Within each set, thepercentage of cell lysis was determined for 5 antibodies at a timerelative to controls I and II at 0, 1, and 6 hrs. Antibody was bound tothe cells by incubating 2 μg of each antibody with 10⁶ Daudi cells for45 min (RT). For the zero time point, the cells were washed andimmediately resuspended in 100 μl of medium containing 20% normal humanserum complement, then incubated for 45 min at 37° C., 5% CO₂. For the 1and 6 hr time points, 10⁶ cells were washed following antibody binding,re-suspended in 12 ml fresh media in a 15 ml Falcon tube, and incubatedat on a mechanical inverter for 1 and 6 hrs, respectively. Cells werewashed at the completion of the selected time points and incubated inmedium containing 20% normal human serum complement, and incubated for45 min. Following serum incubation, 7-amino-actinomycin D (7AAD) wasadded to each sample and incubated for 15 min at RT to assess cellviability. Percent cytotoxicity was determined at each time point bysetting regions as a forward scatter versus 7AAD two-dimensional scatterplot that represented 7AAD positive and negative cells, with debrisexcluded from both regions. Percent cytotoxicity was plotted for eachtime point as 100 minus percentage of 7AAD-negative cells (Table 5-7).

TABLE 5 % Cytotoxicity Antibody 0 hour 1 hour 6 hour Control I 98.5 86.916 Control II 99.6 99.1 98.5 10F2-13 99.6 99.2 98.5 8G6-5 99.6 99.2 97.79D4-7 99.4 99.0 96.0 2B7-7 99.4 99.4 98.3 9C11-14 55.8 22.3 12.9

TABLE 6 % Cytotoxicity Antibody 0 hour 1 hour 6 hour Control I 91.9 65.352.1 Control II 98.3 98.6 97.7 6B9-4 98.1 98.5 97.5 3H7-6 97.5 94.0 67.66F6-1 97.1 97.1 76.4 10E3-17 97 96.2 79.2 9E4-20 67.4 31.2 49.9

TABLE 7 % Cytotoxicity Antibody 0 hour 1 hour 6 hour Control I 98.3 81.120.5 Control II 99.1 99.2 98.7 7E1-13 98.1 98.5 89.3 3B9-10 98.4 97.982.2 9C3-8 98.7 98.5 76.9 9H4-12 43.2 17.4 23.1 2C11-4 29.1 14 22.4

Example 8 Biochemical Off-Rate of the Human Anti-CD20 Antibodies

Biochemical off-rates for selected test anti-CD20 antibodies weredetermined and compared with control antibodies I and II. Two selectedhuman antibodies, control I or II (each 2 μg/ml) were incubated withCD20-expressing Raji cells, at 10⁶/ml, for 2 hrs at RT. The cells werethen washed, excess antibody was removed, re-suspended in 1%serum-containing medium, and incubated at 37° C. At time 0, 15, 30, 45,60, 90, 120, and 180 min, an aliquot of 1 ml of cells was removed,washed, stained with PE-labeled anti-hFc antibody, and FACS analysisconducted. Mean fluorescent intensity (MFI) was used as an indicator ofthe amount of antibody bound to the cell surface. Biochemical off rateswere calculated by setting the percentage binding at time zero as 100%.The experiment was repeated 5 additional times, and biochemical off ratefor 12 the test antibodies determined and compared to control I and II(Tables 8-13).

TABLE 8 Time % Binding (min) Control I Control II 9C11-14 10F2-13 0100.00 100.00 100.00 100.00 15 58.36 69.04 50.86 74.85 30 47.04 72.0342.22 73.99 45 33.77 74.00 28.77 70.77 60 22.96 61.38 17.49 54.30 9011.82 54.43 9.66 51.12 120 6.89 51.33 5.11 47.40 180 2.73 52.73 2.0651.65

TABLE 9 Time % Binding (min) Control I Control II 8G6-5 9D4-7 0 100.00100.00 100.00 100.00 15 67.11 80.54 86.48 81.00 30 51.18 81.20 73.0982.76 45 41.97 85.86 86.95 80.73 60 31.17 85.44 83.93 74.50 90 15.5381.30 73.26 62.59 120 13.08 73.68 67.93 45.99 180 2.42 51.57 47.96 22.34

TABLE 10 Time % Binding (min) Control I Control II 3H7-6 6F6-1 0 100.00100.00 100.00 100.00 15 68.02 90.93 69.38 87.04 30 55.97 84.05 56.5880.86 45 29.49 64.85 33.12 56.45 60 33.24 86.17 36.98 68.75 90 15.4280.84 19.45 60.57 120 9.40 82.08 12.25 54.56 180 3.40 69.25 3.97 34.60

TABLE 11 Time % Binding (min) Control I Control II 2B7-7 6B9-4 0 100.00100.00 100.00 100.00 15 59.47 82.97 88.14 91.05 30 58.96 69.72 90.5395.32 45 49.57 78.71 90.30 96.45 60 30.98 64.19 76.95 79.77 90 18.4167.06 69.17 64.66 120 8.46 58.03 66.95 60.04 180 2.70 51.73 64.01 49.03

TABLE 12 Time % Binding (min) Control I Control II 7E1-13 10E3-17 0100.00 100.00 100.00 100.00 15 73.07 81.04 89.23 88.88 30 51.70 86.2783.34 78.82 45 34.75 87.98 79.89 69.99 60 22.53 76.71 73.89 66.64 9014.01 87.36 96.29 65.20 120 8.54 93.79 94.46 50.86 180 3.44 84.95 89.5829.76

TABLE 13 Time % Binding (min) Control I Control II 3B9-10 9E4-20 0100.00 100.00 100.00 100.00 15 73.84 88.42 77.88 40.59 30 58.06 83.5776.63 15.90 45 39.64 85.97 70.32 6.76 60 26.86 75.25 62.64 3.31 90 12.8967.46 55.16 1.60 120 6.83 61.69 47.74 0.97 180 3.27 68.62 48.25 0.81

Example 9 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay

ADCC induced by selected human anti-CD20 antibodies was assessed usingDaudi cells (cells from a human lymphoma cell line that endogenouslyexpresses CD20). Briefly, Daudi cells (10,000 cells/well in 50 μl) werefirst mixed with an equal volume of serially diluted human anti-CD20antibody, resulting in a final antibody concentration ranging from 0.169pM to 10 nM, and incubated for 10 min at RT in a 96-well plate(control=wells without ab). Separately, human peripheral bloodmononuclear cells (PBMCs, effector cells) were prepared following aconventional Ficoll-Hypaque gradient centrifugation enrichmentprocedure. Enriched PBMCs were collected, washed, and plated in RPMI1640 containing 10% heat inactivated FBS, 2 mM glutamine and 50 nMbeta-mercaptoethanol. The cells were then stimulated with 5 ng/ml humanIL-2 for three days, washed once in media, then used directly in theADCC assay. Approximately 300,000 PBMCs were added to each mixture ofantibody and target cells to give a final ratio of effector to targetcells of approximately 30:1. The 96-well plates were then incubated for4 hr and centrifuged at 250×g. Supernatants were harvested and assayedfor lactate dehydrogenase (LDH) activity using the CYTOTOX 96®Non-Radioactive Cytotoxicity Assay system (Promega) (Table 14).

TABLE 14 Antibody EC₅₀ (pM) N 9C11-14 10.22 4 9E4-20 2.37 3 3B9-10 6.772 8G6-5 14.83 5 10F2-13 6.68 7 6F6-1 5.15 4 7E1-13 2.14 3 9D4-7 1.53 32C11-4 1.45 1 10E3-17 1.20 3 2B7-7 1.99 3 6B9-4 4.27 3 9C3-8 11.02 33H7-6 11.11 3 9H4-12 33.82 1

Example 10 Therapeutic Activities of Anti-CD20 Antibodies with a HumanLymphoma Xenograft Mouse Model

In vivo efficacy studies for selected anti-CD20 antibodies were carriedout using a human non-Hodgkin's B-cell lymphoma xenograft mouse model.Female severe combined immune deficient (SCID) mice were purchased at 6weeks of age. After one week of acclimation, 2.5 million freshlyharvested Raji cells (cells from a human non-Hodgkin's B-cell lymphomacell line) were injected intravenously into each mouse. Each Rajicell-engrafted mouse was then treated with human FC (hFC), control I,control II, 8G6-5, 9D4-7, 10F2-13, or 7E1-13, each at 10 mg/kg, viaintravenous injection through the lateral tail 3, 6, and 9 days afterthe engraftment. Mice were monitored for a period up to 180 days. Miceexhibiting signs of disease including hind-limb paralysis, cachexia, andoccasional large local tumor mass were euthanized by CO₂ asphyxiation.Symptom-free survival curves were constructed using the Kaplan-Meiermethod (FIG. 1). The results are expressed as percent survival as afunction of symptom free survival time. These results show that ab10F2-13 increased survival times significantly in the animal model, fromabout 20 days (hFc control treated animals) to about 180 days (more thana 9-fold increase in survival rate) (50% of treated animals survivedabout 20 days (hFc control), about 40 days (control I), about 85 days(control II), and more than 180 days (10F2-13). These increased survivaltimes are at least 2-fold greater (relative to control II), about4.5-fold greater (relative to control I), or at least about 9-fold orgreater relative to hFc-treated animals.

1. A method for treating a B-cell lymphoma in a human subject, themethod comprising administering to the subject an antibody orantigen-binding fragment thereof that specifically binds human CD20,wherein the antibody or antigen-binding fragment thereof does not bind amutant human CD20 with amino acid changes to SEQ ID NO:1 consisting ofY775, N163D and N166D; and wherein the antibody or antigen-bindingfragment thereof, when administered to a human non-Hodgkin's B-celllymphoma xenograft mouse model, causes 50% of treated animals to survivefor more than 180 days after tumor cell engraftment.
 2. The method ofclaim 1, wherein the human subject has non-Hodgkin's B-cell lymphoma. 3.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof exhibits 4% or lower binding to a mutant human CD20 with aminoacid changes to SEQ ID NO:1 consisting of N163D and N166D, relative to anon-mutated human CD20 (SEQ ID NO:1).
 4. The method of claim 1, whereinthe antibody or antigen-binding fragment thereof exhibits 2% or lowerbinding to a mutant human CD20 with amino acid changes to SEQ ID NO:1consisting of Y77F and N163D, relative to a non-mutated human CD20 (SEQID NO:1).
 5. The method of claim 1, wherein the antibody orantigen-binding fragment thereof exhibits 1% or lower binding to amutant human CD20 with amino acid changes to SEQ ID NO:1 consisting ofY77F and N166D, relative to a non-mutated human CD20 (SEQ ID NO:1). 6.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises: (i) a heavy chain complementarity determining region1 (HCDR1) having the amino acid sequence of SEQ ID NO:341; (ii) a heavychain complementarity determining region 2 (HCDR2) having the amino acidsequence of SEQ ID NO:343; (iii) a heavy chain complementaritydetermining region 3 (HCDR3) having the amino acid sequence of SEQ IDNO:345; (iv) a light chain complementarity determining region 1 (LCDR1)having the amino acid sequence of SEQ ID NO:349; (v) a light chaincomplementarity determining region 2 (LCDR2) having the amino acidsequence of SEQ ID NO:351; and (vi) a light chain complementaritydetermining region 3 (LCDR3) having the amino acid sequence of SEQ IDNO:353.
 7. The method of claim 6, wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable region(HCVR) having the amino acid sequence of SEQ ID NO:339, and a lightchain variable region (LCVR) having the amino acid sequence of SEQ IDNO:347.